Polycyclic pyrimidine -2,4(1H,3H)-diones with functionalized alkyl residues at the 1- and/or 3-position(s); methods for their synthesis and pharmaceutical preparation

ABSTRACT

Polycyclic pyrimidine-2,4(1H,3H)-diones with functionalized alkyl residues at the 1-, the 3-, or both position(s); methods for their synthesis, production, and pharmaceutical preparation. The invention concerns the synthesis of the above-described compounds, their chemical and structural characterization, and the analysis of their physiological/pharmacological activities in vitro and in vivo. These goals have been attained by the specification of routes of synthesis, methods for the production of the compounds, and the presentation of compound-specific characteristics. The substances encompassed by the present invention demonstrate pharmacologically significant collangenase/matrix metalloproteinase inhibitory activities. The specific example 1-(3-mercaptoprop-1-yl)-3-methyl-chinazolin-2,4(1H,3H)-dione will be presented in detail.

This application is a 371 of PCT/EP00/08126, filed Aug. 21, 2000.

DESCRIPTION

The invention concerns polycyclic pyrimidine-2,4(1H,3H)-diones withfunctionalized alkyl residues at the 1- and/or 3-position(s) with thegeneral structures Ia and Ib:

where

-   -   R¹ is a hydrogen, methyl, or ethyl group    -   R² is a hydrogen or methyl group    -   R³ is a mercapto- or hydroxyaminoacylalkylthio-(—SAlkCONHOH)        group    -   Alk is an alkyl group (C₁-C₅, branched or unbranched)    -   R⁴ is a hydrogen, Alk, benzyl, or phenyl group    -   n is 0, 1, or 2    -   Alk* are alkylenes {C₄-C₁₂, unbranched or branched, with the        exception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]}    -   X is a mercapto- or hydroxyaminoacylalkylthio-(—SAlkCONHOH)        group    -   A is an annealed benzene ring and    -   R⁵ is a hydrogen, 6-methyl, 8-methyl, 6-fluoro, 6-chloro,        6-bromo, 6-methylthio, or a 6,7-dimethoxy group, or        -   in the 2,3 positions an annealed thiophene ring, which if            necessary is dimethyl-substituted in the 4,5 positions or            condensed with a cyclopentene, cyclohexene, or cycloheptene            ring,    -   as well as their tautomers and salts, methods for their        synthesis and pharmaceutical preparation, especially regarding        their applications in diseases of humans and animals, by which        the inhibition of collagenases and related metalloproteinases        (matrix metalloproteinases, MMPs) is involved in the healing of        such diseases, or exerts a palliative effect on the pathological        symptoms caused by these enzymes, or interferes with the normal        activity of such enzymes to the benefit of the patient.

Compounds with the general structures Ia and Ib have not yet beendescribed in professional literature. One exception is a compound withthe general structure lb with R⁵═H and Alk*=—CH₂CH₂—CH(CH₃)— and X═SH.This substance is described in EP 0 454 060 as3-(mercapto-3-methylpropyl)-chinazolin-2,4(1H,3H)-dione with a branchedalkyl ═C₄. It was ascribed immunostimulatory and immunorestorativeeffects for applications in disorders of the immune system and in viralinfections.

In DD 293 816, the applicant presents in structure II a chinazoline inwhich n=2 and R²═CH₃, which may be described as3-(mercapto-3-methylpropyl)-chinazolin-2,4(1H,3H)-dione. It served asthe starting compound for the synthesis of3-(alkylthioalkyl)-2,4-dioxo-1,2,3,4-tetrahydrochinazolinene. Similarly,in DD 293 817, methods are described in which compounds shown instructure II with n=2 and R²═CH₃, thus3-(mercapto-3-methylpropyl)-chinazolin-2,4(1H,3H)-dione, serves as asynthetic intermediate.

It is known that the enzymatic activities of metalloproteinases arephysiologically regulated by specific activation and inhibitorymechanisms. An uncontrolled enzymatic activation or insufficientinhibition of these enzymes, especially in the case of rheumaticdiseases, results in dysregulated cartilage degradation and subsequentchronic and painful pathological changes in the affected articulations.

Another example of the pathological effects of collagenases/MMPs istumor metastasis and invasion. Secreted collagenases/MMPs degrade densecollagenous connective tissue, allowing the cancerous cells to migrateaway from the tumor, to gain access to the circulatory or lymphaticsystem, to exit that circulation, and to establish metastases in organsdistant from the site of the original tumor.

A further medical/cosmetic example of the effects of dysregulatedcollagenase activity is UV-induced erythema, most commonly induced byexcessive/intensive solar irradiation (i.e. sunburn). Collagenases/MMPsin the exposed skin are activated by the UV radiation in sunlight orfrom tanning devices; these activated enzymes degrade the connectivetissue collagen of the skin and blood capillaries, leading to thewell-known symptoms of sunburn.

The pathological effects caused by the activities of collagenases/MMPsdescribed in the preceding examples can be prevented by stableinhibitors of these enzymes. It is further likely that specificinhibitors will be able to block only the targeted enzyme, allowing thepossibility to interrupt the pathological cartilage destructioncharacteristic of rheumatoid arthritis with minimal side effects.

Methods for the preparation of compounds with collagenase inhibitoryeffects have been described. As a rule, these active substances have apeptide backbone based on the structures of natural inhibitors orsubstrates. These peptide- or pseudopeptide substrate analogues possessan essential zinc-binding group, which chelates the active-site zincatom in the metal-dependent collagenases/MMPs.

For therapeutic applications, peptide analogue inhibitors have manydisadvantages, including inadequate uptake, short biological half-lives,and only limited stability in vivo.

Thus, there is a dear need to develop nonpeptide pharmaceuticalinhibitors that lack the above-described disadvantages of thepeptide-based collagenase/MMP inhibitors. These newcollagenase-inhibiting substances must be biologically stable, readilyabsorbed, and have a high and specific affinity for the zinc atom in thecollagenase/MMP active site.

The purpose of this invention is to discover new nonpeptide chemicalsubstances that possess collagenase/MMP inhibitory activity. It is thefurther purpose of this invention to develop methods for both theproduction of such inhibitory substances and the development ofpharmaceuticals that contain these substances.

These goals have been suitably achieved. The dependent claims concernadvantageous procedural variations of the invention.

The polycyclic pyrimidine-2,4(1H,3H)-diones with functionalized alkylresidues in the 1- and/or 3-posibon described in the invention have thegeneral structures la and lb,

where

-   -   R¹ is a hydrogen, methyl, or ethyl group    -   R² is a hydrogen or methyl group    -   R³ is a mercapto- or hydroxyaminoacylalkylthio-(—SAlkCONHOH)        group    -   Alk is an alkyl group (C₁-C₅, branched or unbranched)    -   R⁴ is a hydrogen, Alk, benzyl, or phenyl group    -   n is 0, 1, or 2    -   Alk* are alkylenes {C₄-C₁₂, unbranched or branched, with the        exception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]}    -   X is a mercapto- or hydroxyaminoacylalkylthio-(—SAlkCONHOH)        group    -   A is an annealed benzene ring and    -   R⁵ is a hydrogen, 6-methyl, 8-methyl, 6-fluoro, 6-chloro,        6-bromo, 6-methylthio, or a 6,7-dimethoxy group,    -   or        -   in the 2,3 positions an annealed thiophene ring, which if            necessary is dimethyl-substituted in the 4,5 positions or            condensed with a cyclopentene, cyclohexene, or cycloheptene            ring.

The invention explicitly concerns the core compounds described in theinvention, their tautomers and their salts, particularly those saltsmost appropriate for pharmacological applications such as sodium andammonium.

The compounds described by structures la and lb are novel chemicalsubstances with enzyme inhibitory, specifically collagenase/MMPinhibitory qualities, which allows them to be applied to great advantagein human and veterinary medicine. Furthermore, these novel substancesreadily lend themselves to further chemical modification to producecompounds with analogous or altered spectra of activity.

Of the many compounds with the general structures la and lb, specialemphasis is placed on the development of compounds with structures IIaand IIb:

where R¹, R², R³, R⁴, Alk, Alk*, n, and X are defined as above.

Comprised in the present invention are compounds with the generalstructures Ia and Ib either as such or as their tautomers, or as theirsalts, particularly pharmaceutically harmless alkali or ammonium salts.

The methods described in the invention for the preparation of thecompounds with the general structures Ia, where R³ and R⁴ are a mercaptogroup and a hydrogen, respectively, are characterized by the followingprocedure:

-   -   (A) the reaction of 2-(alkenylaminoy)-1-carboxylic acid amide        with the general structure III,        where R⁶ is an alkenyl group (C₃-C₆) such as allyl, methallyl,        crotyl, 1-butene-4-yl, 3-pentene-1-yl, or 3-hexene-1-yl, and A        and R⁵ are defined as above, with a reagent providing a (—C═S—)        structural element such as thiophosgene, thiourea, ammonium or        alkalithiocyanate/HCl, 1,1′-thiocarbonylbisimidazole, or        benzoylisothiocyanate in a polar, aprotic solvent.    -   stir the reaction mixture    -   remove solvent under vacuum    -   add dilute alkali solution and gently heat to circa 60° C.,    -   separate the insoluble fraction by filtration,    -   cool and acidify the filtrate,    -   heat the resulting compound with the general structure IV,        where A, R⁵, and R⁶ are as defined above,    -   with centrated mineral acid, such as hydrochloric acid,        hydrobromic acid and/or sulfuric acid, or a mixture of these        mineral acids with glacial acetic acid and/or formic acid to        reflux, cool the reaction mixture,    -   dry the resulting compound with the general structure V,        where R⁷, R⁸, R⁹, and R¹⁰ are hydrogen, a methyl, or an ethyl        group and A and R⁵ are as defined above,    -   or respectively, dry the resulting compound with the general        structure VI,        where R¹¹, R¹², and R¹³ are a hydrogen, a methyl, or an ethyl        group, and A and R⁵ are as defined above,    -   in a vacuum desiccator over potassium hydroxide or    -   stir the above compound in a dilute, aqueous sodium carbonate        solution,    -   isolate the compound,    -   warm the compound in dilute mineral acid, such as hydrochloric        acid, hydrobromic acid and/or sulfuric acid, or a mixture of        these mineral acids with glacial acetic acid and/or formic acid        to reflux,    -   cool the reaction mixture,    -   wash and dry the crystallized material, yielding the compound        with the general structure la or its tautomers, in which R⁴ is        without exception hydrogen.

The method described in the invention for the preparation of thecompounds with the general structure la, where R³ is a mercapto group,R⁴ is a hydrogen, benzyl, or phenyl group, n=1 or 2, and Alk, A, and R⁵are as defined above, is characterized by the following procedure:

-   B) the reaction of bi- and tricyclic 3-alkyl (resp. benzyl or    phenyl)-pyrimidine-4(3H)-on-2(1H)-thionene with the general    structure VII,    where R4 is a hydrogen, an alkyl, a benzyl or a phenyl group, and A    and R⁵ are defined as above,    with 1, ω-dihalogenalkanene with the general structure VIII,    Hal(CH₂)_(m)Hal  (VIII)    where m=2, 3, or 4, and Hal is chlorine, bromine, or iodine,    -   In an aprotic dipolar solvent, preferably in dimethylformamide        with the addition of potassium carbonate with gentle stirring at        room temperature,    -   add dilute hydrochloric acid,    -   heat until reflux,    -   filter the hot reaction mixture,    -   cool and store the solution at 4° C.,    -   yielding the compound with the general structure Ia,        where R³ is a mercapto, R⁴ a hydrogen, a benzyl, or a phenyl        group, and A and R⁵ are as defined above, or-   C) the reaction of 2-ammonium carboxylic acid methyl ester    thiocyanate with the general structure IX,    where A and R⁵ are as defined above,    with 1, ω-dihalogenalkanene with the general structure VIII,    where m=2 or 3 and Hal is chlorine, bromine, or iodine,    -   heat the reactants with stirring until reflux    -   cool and isolate the precipitate,    -   wash the precipitate with diethylether and dry,    -   dissolve the precipitate in water,    -   filter the solution and add dilute aqueous NaOH to pH 10,    -   isolate the precipitate and wash with water,    -   dry the precipitate with vigorous shaking with chloroform,    -   isolate the precipitate and dry.    -   Recrystallize from solvent, preferably 2-methoxyethanol        yielding compounds of the general structure V,        obtained by reaction with 1,3-dibromomethane,        where R⁷, R⁸, R⁹, and R¹⁰ are hydrogen and A and R⁵ are as        defined above,        or the compound of the general structure VI,        obtained by reaction with 1,3-dibromopropane        where R¹¹, R¹², and R¹³ are hydrogen, and A and R⁵ are as        defined above.

Heat the compounds of the general structures V or VI in dilute mineralacid, such as hydrochloric acid, hydrobromic acid and/or sulfuric acid,or a mixture of these mineral acids with glacial acetic acid and/orformic acid to reflux,

-   -   cool the reaction mixture,    -   wash and dry the crystallized material to yield a compound of        the general structure Ia or its tautomers,        where R¹, R², and R⁴ are hydrogens, R³ is a mercapto group, n=1        or 2, and A and R⁵ are as defined above.

The methods described in the invention for the preparation of thecompounds with the general structure 1b, where Alk* is n-butylene(—CH₂—CH₂—CH₂CH₂—), X is a mercapto group, and A and R⁵ are as definedabove, are characterized by the following procedures:

-   D) React 2-isothiocyanato-1-carboxylic acid ester of the general    structure X,    where Alk, A, and R⁵ are defined as above,    with 4-aminobutan-1-ol at room temperature with vigorous and    prolonged stirring    -   add water to obtain the compounds of the general structure XI,        where A and R⁵ are as defined above,    -   react the compounds of the general structure XI with        concentrated mineral acid, such as hydrochloric acid,        hydrobromic acid and/or sulfuric acid, or a mixture of these        mineral acids with glacial acetic acid and/or formic acid to        reflux,    -   cool the reaction mixture and isolate the crystallized material,    -   add aqueous sodium carbonate to the crystals to obtain ca. pH 9,    -   isolate the crystallized material, wash with water, and dry to        obtain the polycyclic        2,3,4,5-tetrahydro-7H-[1,3]thiazepino[2,3-a]pyrimidin-7-one of        the general structure XII,        where A and R⁵ are as defined above.

Heat the compounds of the general structures XII in very dilute mineralacid, such as hydrochloric acid, hydrobromic acid and/or sulfuric acid,or a mixture of these mineral acids with glacial acetic acid and/orformic acid to reflux,

-   -   cool the reaction mixture,    -   wash and dry the crystallized material to obtain compounds of        the general structure Ib or its tautomers where    -   Alk* is n-butylene (—CH₂—CH₂—CH₂—CH₂—), X is a mercapto group,        and A and R⁵ are as defined above.

The methods described in the invention for the preparation of thecompounds with the general structure 1b, where Alk* is alkylene (C₄-C₁₂;unbranched and branched, with the excepton of 3-methylpropylene[—CH₂—CH₂—CH(CH₃)—], X is a mercapto group, and A and R⁵ are as definedabove, are characterized by the following procedures:

-   -   E) React 2-alkoxycarbonylamino-1-carboxylic acid alkyl ester of        the general structure XIII,        where Alk is an alkyl group (C₁-C₃) and A and R⁵ are as defined        above, with aminoalkanols of the general structure XIV,        where Alk* is alkylene {C₄-C₁₂; unbranched and branched, with        the exception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]}by        warming in a well-known reaction,    -   cool the reaction mixture,    -   add water and dilute hydrochloric acid to ca. pH 4,    -   isolate the precipitate, wash with water and dry to yield the        polycyclic 3-(ωhydroxyalkyl)-pyrimidine-2,4(1H,3H)-diones with        the general structure XV,        where Alk* is alkylene {C₄-C₁₂; unbranched and branched, with        the exception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]} and A        and R⁵ are as defined above.

React the compounds with the general structure XV with concentratedhydrochloric acid, phosphorus trichloride, or phosphorus oxide chloride;preferably, however, with hydrobromic acid:

-   -   heat to reflux,    -   cool the reaction mixture,    -   isolate the precipitate, wash with water, and dry to yield the        polycyclic 3-(ω-haloalkyl)-pyrimidine-2,4(1H,3H)-diones with the        general structure XVI,        where Hal is chlorine, or bromine, and Alk*, A, and R⁵ are as        defined above.

React the compounds of the general structure XVI in boiling polarsolvent with thiourea:

-   -   heat to reflux,    -   cool the reaction mixture,    -   add water and dilute sodium hydroxide to obtain an alkaline pH        value,    -   after clearing the solution by filtration,    -   add dilute hydrochloric acid to ca. pH 3,0,    -   isolate the precipitate, what with water and dry to yield        compounds with the general structure Ib and its tautomers        where Alk* is alkylene {C₄-C₁₂; unbranched and branched, with        the exception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]}, X is a        mercapto group, and A and R⁵ are as defined above.

The methods described in the invention for the preparation of compoundswith the general structures 1a or 1b, where R³ or X is ahydroxyaminoacylalkylthio (—SAlkCONHOH) group and R¹, R², R⁴, Alk, n,Alk*,A, and R⁵ are as defined above, are characterized by the followingprocedures:

-   F) React compounds with the general structures Ia or Ib,    where R³ is a mercapto group, and R¹, R², R⁴, Alk, n, Alk*,A, and R⁵    are as defined above, with N-hydroxyhalogen carboxylic add amides,    preferably 2-choro-N-hydroxyacetamide (ClCH₂CONHOH),    -   in pyridine    -   or in acetone solution, preferably in the presence of a base,    -   at room temperature,    -   distill away the solvent,    -   add water,    -   isolate the precipitate, wash with water and dry to yield        compounds of the general structure Ia or Ib or their tautomers,        where R³ or X is a hydroxyaminoacylalkylthio group.

According to the present invention, the procedures designated (A-H) maybe modified.

For example, further procedures regarding the invention described inmethod (A) include:

-   -   the reaction of the compounds with the general structure III        with benzoylisothiocanate in absolute acetone,    -   in the execution of method (B), another procedure included in        the invention is    -   that the reaction of compounds of the general structure VII with        compounds of the general structure VIII may be carried out in        absolute dimethylformamide in the presence of dry potassium        carbonate.        Studies of Collagenase/MMP Inhibitory Effects

TABLE 1 Inhibition of human matrix metalloproteinases and Clostridiumhistolyticum collagenase by compounds based on the general structures Iaand Ib Lead MMP-2 MMP-8 MMP-9 C. histolyticum compound (50% inhibition)(K_(i) value) (50% inhibition) (50% inhibition) Example 1 (Ia) 23.3 μM33.3 μM 21.0 μM

Example 2 (Ia) 142.9 μM  negative

Example 3 (Ia) 13.2 μM 38.5 μM negative 27.5 μM

Example 4 (Ia)  5.0 μM  7.0 μM  6.0 μM 53.8 μM

Example 5 (Ia) 61.3 μM 70.0 μM 12.5 μM 39.2 μM

Example 6 (Ia) 97.1 μM negative 13.1 μM

Example 7 (Ia)  6.0 μM negative 71.4 μM 11.5 μM

Example 8 (Ia) negative 39.4 μM

Example 9 (Ia) 13.9 μM 43.4 μM

Example 10 (Ia)  6.3 μM 31.5 μM

Example 11 (Ia) 33.3 μM 22.4 μM

Example 12 (Ib)  8.3 μM   11 μM   20 μM

Example 13 (Ib)   20 μM

Example 14 (Ia)   20 μM negative   50 μM

Example 17 (Ia)   20 μM negative   10 μM

Example 18 (Ib)  8.3 μM  6.0 μM   10 μM

Example 19 (Ib)  7.1 μM  3.0 μM

Example 20 (Ia) 71.4 μM

Inhibition of Clostridium histolyticum Collagenase (EC 3.4.24.3)

The activity of Clostidium histolyticum collagenase may be readilydetermined by a simple spectrophotometric assay using the syntheticchromogenic peptide substrateN-(3-[2-furyl]acryloyi)-leucine-glycine-proline-alanine (FALGPA; VanHart, H. E. and Steinbrink, D. R. 1981, Anal. Biochem. 113, 356). Thedegree of inhibition of the degradation of this chromogenic substrate bythe addition of the various test substances described in Table 1 may beeasily quantified. The enzyme and substrate are commercially available.

Inhibition of Human Matrix Metalloproteinase-2 (MMP-2; gelatinase A)

MMP-2 is easily prepared by standard procedures from the culture mediumof cultivated human fibroblasts in reasonable quantities, and istherefore readily available. The inactive secreted pro-form (pro-MMP-2)can be activated by the addition of trypsin or by the addition oforganomercurial compounds to prepare the enzymatically active matureform of the enzyme. A screening system based on the measurement of theproteolytic activity of MMP-2 was developed to analyse the inhibitoryeffects of the various inhibitors described in Table 1.

To this end, human dermal fibroblasts were obtained and maintained inculture using standard methods. The cell-free culture supernatantcontaining the secreted pro-MMP-2 was treated with trypsin. The trypsinwas inactivated with the specific active-site serine proteinaseinhibitor Nα-p-tosyl-L-lysine-chloromethylketone (TLCK), and the activeMMP-2 was partially purified by gel filtration on Sepharose 4B. Theisolated MMP-2 was identified and characterized by the use of acommercially available immunoassay.

A simple nonradioactive assay was developed to measure MMP-2 activityusing gelatin as a substrate that allows both a large number of samplesto be analysed in parallel and to generate kinetic data. Partiallypurified MMP-2 was incubated with a suitable dilution of gelatin understandardized conditions (time, buffer, pH, temperature, inhibitor,additives), and uncleaved gelatin was separated from degradationproducts by suction filtration through a nitrocellulose filter.High-molecular-weight gelatin is thereby bound to the nitrocellulosefilter, and the low-molecular-weight gelatin-derived peptides passthrough it. The nitrocellulose-bound gelatin can be stained with PonceauS or Commassie Brilliant Blue protein stains, and the degree of stainingquantified densitometrically using a commercially availableelectrophoresis gel documentation system supported by dedicatedanalytical software. The nitrocellulose filtration is conducted using acommercially available dot- or slot-blotting apparatus.

The gelatinase assay described here has been validated by comparisonwith results obtained with an established collagenase assay usingradioactive or fluorescently labeled collagen as substrate.

Preparation of Human Matrix Metalloproteinase-9 (MMP-9; Gelatinase B)

Native MMP-9 was reproducibly obtained with reasonable yield and purityfrom human buffy coat. Buffy coat, to which was added an appropriatevolume of 10% (v/v) Triton-X-100 to obtain a final concentration of 0,4%detergent, was shaken on ice for 30 min; then, one volume oftwofold-concentrated binding buffer (40 mM Tris-HCl pH 7,5, 10 mM CaCl₂,1 M NaCl, 0,2% (v/v) Triton X-100 was added and the mixture shaken onice for a further 30 min.

The mixture was then centrifuged for 15 min at 16,000 rpm (30,590×g) ina Kendro SS-34 rotor at 4° C. and the resulting supernatant filteredover glass wool. The filtrate was incubated batchwise withgelatin-agarose equilibrated in binding buffer for 1 h on ice withcontinuous shaking The MMP-9-bound gelatin-agarose was then loaded intoa chromatographic tube and the column washed with at least ten bedvolumes of binding buffer, or until no protein was detected byabsorbance at 280 nm in the wash buffer. MMP-9 was eluted from thegelatin affinity column by application of two bed volumes of bindingbuffer containing 5% (v/v) dimethylsulfoxide (DMSO).

The eluate was applied to a gel filtration column of Sephadex G-75 bothto exchange buffer and to eliminate small amounts of contaminatingMMP-2. The G-75 column was prepared and eluted with Buffer I [20 mMTris-HCl pH 7,5, 5 mM CaCl₂, 100 mM NaCl, 0,1% (v/v) Triton X-100].

The eluted MMP-9 includes the three characterized forms of the enzyme:monomer, homodimer, and heterodimer (complex with lipokalin). The purityof MMP-9 was estimated to be ca. 90%, with the remaining contaminatingprotein being fibronectin and small amounts of TIMPS.

The latent MMP-9 was activated by a 30-60 min incubation at 37° C. with1/100 vol trypsin (10 mg ml⁻¹). The trypsin was subsequently inactivatedby addition of the serine proteinase inhibitorphenlymethylsulfonylfluoride (PMSF) or the more specific trypsininhibitor TLCK.

Cloning of the Gene and Expression of the Catalytic Domain of HumanMatrix Metalloproteinase-8 (MMP4; Neutrophil Collagenase)

The catalytic domain of MMP-8 was chosen as a further test enzymebecause of its high stability and constitutive activity, obviating theneed for enzymatic or chemical activation, which is a well-known sourceof error, e.g. the commonly used organomercurial compounds used toactivate pro-MMPs may interfere in subsequent assays or affect theactivity of the enzyme, leading to erroneous interpretation of results.The cloning strategy was designed to express heterologously only thecatalytic domain, rather than the entire MMP-8, in Escherichia coli.With the so-constructed expressed catalytic domain, we obtained a stableenzymatically active, highly pure enzyme that is well suited for routineinvestigation of the efficacy of the synthetic inhibitors and provideshighly reproducible test results.

The cloning and expression of the recombinant catalytic domain of MMP-8follows the protocol developed by Schnierer et al. (Schnierer, S.,Kleine, T., Gote, T., Hilleman, A., Knäuper, V., and Tschesche, H.,1993, The recombinant catalytic domain of human neutrophil collagenaselacks type I collagen substrate specificity, Biochem. Biophys. Res.Commun. 191, 319-326).

Quantitative Fluorimetric Assay for Matrix Metalloproteinases

The enzymatic cleavage of commercially available synthetic internallyquenched fluorigenic peptide substrates such as7-methylcoumarin-4-yl-acetyl-prolyleucyl-glycyl-leucyl-(dinitrophenyl-L-α,β-diaminopropionyl)-alanyl-arginylamide(Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH₂) at the scissile-Gly-Leu-peptide bond separates the fluorescent Mca group from thedinitrophenol quencher, resulting in a strong linear increase influorescence intensity that can be readily measured in a fluorimeter(λ_(exitation)328 nm, λ_(emission) 393 nm) over the first few minutes ofthe reaction. The specificity of the assay for matrix metalloproteinasesis provided by the type I collagen-derived sequence -Pro-Leu-Gly-Leu- inthe synthetic substrate, as well as by the incubation conditions. Totest the efficacy of the synthetic collagenase/MMP inhibitors, theresidual enzymatic activity remaining after an incubation of enzyme plusdifferent amounts of inhibitor is measured at a fixed enzyme andsubstrate concentration. For each inhibitor, three test series atdifferent fixed substrate concentrations were carried out. From thetime-dependent increase in fluorescence intensity measured, the velocity(v) of the enzymatic activity can be calculated as increase in arbitraryfluorescence intensity units per minute. The inhibition constant Ki iscalculated graphically by the method of Dixon (1953) by plotting thereciprocal of the reaction velocity 1/v on the ordinate, and theinhibitor concentration on the abscissa. The x-intercept of the linearplot is defined as minus K₁ value.

Assay

Materials

1984 μl assay buffer (100 mM Tris-HCl pH 7,5, 100 mM NaCl, 10 mM CaCl₂,0,05% Brij 35)

-   -   2 μl inhibitor dissolved in DMSO, or DMSO alone as a solvent        control    -   4 μl enzyme (MMP-2, MMP-9, or the catalytic domain of MMP-8)    -   10 μl Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH₂ in DMSO        Method

-   1. Incubate buffer, enzyme, and inhibitor for 5 min at room    temperature, with stirring.

-   2. Start the reaction by adding 10 μl substrate in DMSO

-   3. Record the increase in fluorescence intensity over 2 min.    Confirmation of the Inhibitory Activity by Protein Zymographic    Analysis

Zymography permits the rapid and simple analysis of complex mixtures ofproteinases. A protease substrate, usually a protein like casein,fibrinogen, or gelatin, is co-polymerized in a polyacrylamide gel andthe enzyme mixture to be analysed is electrophoretically separated inthe gel. After the electrophoresis is complete, the gel is incubated ina suitable buffer to allow the separated proteolytic enzymes to digestthe immobilized substrate. After fixation and staining, colorless bandsappear on a stained substrate background; the dear bands indicate theposition on the gel where the separated proteinases have digested thesubstrate.

The compounds found to be effective collagenase inhibitors byfluorimetric analysis were analysed on gelatin-containing polyacrylamidegels. The potential inhibitors may either be co-polymerised in the gelwith the gelatin substrate or applied with the enzyme after a suitableincubation prior to electrophoresis. This is possible because the testcompounds, in contrast to the collagenases/MMPs, are not charged (belowthe pK value of the —SH group) and therefore do not migrate alone duringthe electrophoresis.

By this method, crude samples containing collagenase/MMP activity can beanalysed. The effects of the novel compounds on collagenase/MMPactivities in cell culture supernatant, synovial fluid, and tissueextracts can be tested by zymographic analysis. Metalloproteinases canbe identified in complex mixtures containing other proteolytic enzymesby inhibition with the zinc chelator 1,10-phenanthroline (always usingthe same concentration of the nonchelating analogue 1,7-phenanthrolineas a specificity control).

The invention will be illustrated in detail in the following specificexamples:

EXAMPLE 1

(R,S)-1-mercaptopropyl)-chinazolin-2,4(1H,3H)-dione

(structure 1a, R¹=hydrogen, R²=methyl, R³=mercapto, R⁴=hydrogen, n=1,A=benzene ring with R⁵=hydrogen)

-   a) 2 allylaminobenzoic acid amide (structure III, R⁶=allyl,    A=benzene ring with R⁵=hydrogen)

2-aminobenzamide (13,60 g; 0,1 mol) is suspended in 80 ml water, towhich is added 6,90 g potassium carbonate and 7,70 g (0,1 mol)allylchloride. The reaction mixture is then refluxed for 2 h. Aftercooling, crystals are precipitated from ehtanol/water (1:1, v/v).Between 13,8 and 14,3 g colourless crystals of 2-allylbenzoic acid amideare obtained.

-   Yield 79%.-   C₁₀H₁₂N₂O (176,2).-   F.: 88°-92° C. (ethanol/water).-   b) 1-allylchinazolin-4(3H)-on-2-(1H)-thione (structure IV, R⁶=allyl,    A=benzene ring with R⁵=hydrogen)

The thin-layer chromatographically-pure compound described in (a) (8,81g; 0,05 mol) is dissolved in 50 ml acetone and reacted with 0,06 molbenzoytisothiocyanate. The reaction mixture is stirred at roomtemperature for 24 h, after which the solvent is removed under vacuum.With constant stirring, 50 ml water is added to the residue, and then10% aqueous sodium hydroxide is added to pH 9. With further stirring,the suspension is heated slowly to 60° C. and the small amount ofinsoluble material is eliminated by filtration. After cooling, thesolution is acidified with 10% aqueous hydrochloric acid. Theprecipitate is collected by filtration, washed with water, and afterdrying, recrystallized to obtain 5,35 light yellow crystals of1-allylchinazolin-4(3H)-on-2(1H)-thione.

-   Yield 49%.-   C₁₁H₁₀N₂OS (218,3).-   F.:179°180° C. (ethanol).-   IR (νin cm⁻¹): 1608 (NH), 1692 (C═O), 2948 (CH₂).-   MS m/e (% B): M⁺218(27), 203(100), 119(21), 77(33).-   c) (R,S)-2-methyl-1,2-dihydro-5H-thiazolo[3,2-a]chinazolin-5-one    hydrobromide (structure V, R⁷=R⁸=R¹⁰=hydrogen, R⁹=methyl, A=benzene    ring with R⁵=hydrogen)

The thin-layer chromatographically-pure compound described in (b) (2,18g; 0,01 mol) is refluxed in 20 ml concentrated hydrobromic add for 30min. After cooling, the precipitate retained on a glass frit is dried ina vacuum desiccator over potassium hydroxide. The final product obtainedis 1,67 g thin-layer chromatographically-pure colourless crystals of(R,S)-2-methyl-1,2-dihydro-5H-thiazolo[3,2-a]chinazolin-5-onehydrobromide.

-   C₁₁H₁₀N₂OSxHBr (298,3+xHBr).-   F.: 242°247° C. (ethanol).-   IR (ν in cm⁻¹): 1618 (C═N), 1686 (C═O), 2922 (CH₂).-   MS m/e (% B): M⁺218(100), 203(13), 190(49), 175(17).-   d) (R,S)-1-(2-mercaptopropyly)-chinazolin-2,4(1H,3H)-dione    (structure 1a, R¹=hydrogen, R²=methyl, R³=mercapto, R⁴=hydrogen,    n=1, A=benzene ring with R⁵=hydrogen)

The compound described in (c) (1,50 g) is refluxed in a mixture of 0,6ml concentrated sulfuric acid, 1,5 ml glacial acetic acid, and 66 mlwater for 8 h. After cooling, the precipitate obtained on a glass fritis washed with a small amount of water, and after drying, recrystallizedto yield 1,05 g colorless crystals of(R,S)-1-(2-mercaptopropyl)-chinazolin-2,4(1H,3H)-dione.

-   CH₁₁H₁₂N₂O₂S (236,3).-   F.: 170°-173° C. (ethanol).-   IR (ν in cm⁻¹): 1608 (NH), 1682 (C═O), 1702 (C═O), 2554 (SH), 2922    (CH₂).-   MS m/e (% B): M⁺236(6), 204(21), 176(17), 162(89), 149(34),    132(100).-   UV_(ethanol)(in nm) (log E): 220,2 (4,83), 244,2 (4,22), 310,8    (3,94).

EXAMPLE 2

1-(2-mercapto-2-methylpropyl)-chinazolin-2,4(1H,3H)-dione

(structure 1a, R¹=R²=methyl, R³=mercapto, R⁴=hydrogen, n=1, A=benzenering with R⁵=hydrogen).

-   Colorless crystals.-   Yield 76% based on the last step of the synthesis.-   C₂H₁₄N₂O₂S (250,3).-   F.: 183°186° C. (ethanol).-   IR (ν in cm⁻¹): 1610 (NH), 1684 (C═O), 1712 (C═O), 2552 (SH), 2928    (CH₂).-   MS m/e (% B): M⁺+1 251(100), M⁺250(8), 217(9), 176(58), 162(33),    149(100), 132(50).-   UV_(ethanol)(in nm) (log ε): 220,0 (4,36), 245,2 (3,80), 312,2    (3,61).

The preparation of this mercapto derivative is analogous to thatdescribed in Example 1; nevertheless the following intermediates wereisolated and characterized:

-   a) 2-methallylamino benzoic acid amide (structure III, R⁶ methallyl,    A=benzene ring with R⁵=hydrogen).    Prepared from 2-aminobenzamide and methallylchloride.-   Colorless crystals.-   Yield 87%.-   C₁₁H₁₄N₂O (190,2).-   F.: 130° C. (ethanol/water).-   IR (νin cm⁻): 1622 asymm.(NH, C═O), 2910 (CH₂).-   MS m/e (% B): M⁺190(50), 173(86), 158(19), 144(47), 132(100).-   b) 1-methallylchinazolin-4(3H)-on-2(1 H)-thione (structure IV,    R⁶=methallyl, A=benzene ring with R⁵=hydrogen).

Prepared from 2-methallylamino benzoic acid amide andbenzylisothiocyanate in absolute acetone solution.

-   Light yellow crystals.-   Yield 53%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 181°-182° C. (ethanol).-   IR (νin cm⁻¹): 1606 (NH), 1682 (C═O), 2930 (CH₂).-   MS m/e (% B): M⁺²³²(31), 217(100), 199(27), 184(11), 119(13).-   c) 2,2-dimethyl-1,2-dihydro-5H-thiazolo[3,2-a]chinazolin-5-one    (structure V, R⁷=R⁸=hydrogen, R9=R¹⁰=methyl, A=benzene ring with    R⁵=hydrogen).

Prepared from 1-methallylchinazolin-4(3H)-on-2(1H)-thione andconcentrated hydrobromic acid. The precipitate obtained after cooling isresuspended in 5% aqueous sodium carbonate at room temperature.

-   Colorless crystals.-   Yield 87%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 269° C. (ethanol).-   IR (νin cm⁻¹): 1618 (NH), 1684 (C═O), 2923 (CH₂).-   MS m/e (% B): M⁺232(100), 217(15), 204(18), 189(38), 171(26).-   UV_(ethanol)(in nm) (log ε): 234,0 (4,31), 254,5 (4,38), 309,5    (3,90).

The reaction of the compound obtained in step (c) analogous to themethod described under (1d) yields1-(2-mercapto-2-methyl-propylychinazolin-2,4(1H,3H)-dione.

EXAMPLE 3

(R,S)-1-(3-mercaptobut-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure 1a, R¹=R⁴=hydrogen, R²=mercapto, R³=methyl, A=benzene ringwith R⁵=hydrogen).

-   Colorless crystals.-   Yield 86% based on the last step of the synthesis.-   C₁₂H₁₄N₂O₂S (250,3).-   F.: 142°143° C. (ethanol).-   IR (ν in cm⁻): 1610 (NH), 1680 (C═O), 1694 (C═O), 2554 (SH).-   MS m/e (% B): M⁺+1 251(100).

The preparation of this compound is analogous to Example 1;nevertheless, the following intermediates were isolated andcharacterized:

-   a) 2-(but-1-en-4-yl-amino)-benzoic acid amide [structure III,    R⁶=—(CH₂)₂CH═CH₂, A=benzene ring with R⁵=hydrogen].

Prepared from 2-aminobenzamide and 4-bromo-1-butene.

-   Colorless crystals.-   Yield 59%.-   C₁₁H₁₄N₂O (190,2).-   F.: 114°-115° C. (ethanol).-   IR (ν in cm⁻¹): 1622 (C═O), 2930 (CH₂), 2942 (CH₂).-   MS m/e (% B): M⁺190(15), 149(46), 132(100).-   b) 1-(but-en-4-yl)-chinazolin-4(3-on-2(1H)-thione [structure IV,    R⁶=(CH₂)₂CH═CH₂. A=benzene ring with R⁵=hydrogen].

Prepared from 2-(but-1-en-4-yl-amino)-benzoic acid amide andbenzylisothiocyanate in absolute acetone solution.

-   Light yellow crystals.-   Yield 39%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 184°-185° C. (ethanol).-   IR (ν in cm⁻¹): 1608 (NH), 1682 (C═O), 2950 (CH₂).-   MS m/e (%B): M⁺232(35), 203(74), 178(53), 162(8), 145(12), 132(29),    120(100).-   c)    (R,S)-3-methyl-2,3-dihydro-1H,6H-[1,3]thiazino[3,2-a]chinzolin-6-one    (structure VI, R¹¹=R¹²=hydrogen, R13=methyl, A=benzene ring with    R⁵=hydrogen).

Prepared from 1-(but-en-4-yl)-chinazolin-4(3H)-on-2(1H)-thione andconcentrated hydrobromic acid.

After cooling the reaction mixture, the resulting precipitate isresuspended in 5% sodium carbonate at room temperature, filtrated anddried.

-   Colorless crystals.-   Yield 84%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 198 -201° C. (ethanol).-   IR (ν in cm⁻¹): 1636 (C═N), 1708 (C═O), 2926 (CH₂), 2962 (CH₂).-   MS m/e (% B): M⁺232(100), 204(77), 162(36), 132(46).-   UV_(ethanol) (in nm) (log ε): 230,5 (4,27), 257,5 (4,46), 305,5    (3,94).

The reaction of the compound obtained in step (c) analogous to themethod described under (1d) yields(R,S)-1-(3-mercaptobut-1-yl)-chinazolin-2,4(1H,3H)-dione.

EXAMPLE 4

1-(3-mercaptopropyl)-chinazolin-2,4(1H,3H)-dione

(structure 1a, R1=R2=R4=hydrogen, R3=mercapto, n=2, A=benzene ring withR⁵=hydrogen).

-   a) 2,3-dihydro-1H,6H-[1,3]thiazino[3,2-a]chinazolin-6-one (structure    VI, R¹¹=R¹²=R¹³=hydrogen, A=benzene ring with R⁵=hydrogen).

2-ammoniumbenzoic acid methyl ester thiocyanate (5,04 g; 24 mmol)(structure IX, A=benzene ring with R⁵=hydrogen) is refluxed with 50,0 g1,2-dibromopropane with constant stirring for 150 min. After cooling thereaction mixture, the precipitate is collected, washed withdiethylether, and dried. The crude product is dissolved in water and thesolution filtered. The filtrate is adjusted to pH 10 with 10% aqueoussodium hydroxide and resulting precipitate collected, washed with water,and dried. The resulting solid is extensively shaken with 30 mlchloroform. The insoluble material is isolated, dried, andrecrystallized.

-   Colorless crystals.-   Yield 27% based on 2-ammoniumbenzoic acid methyl ester thiocyanate.-   C₁₁H₁₁N₂OS (218,3).-   F.: 236° C. (2-methoxyethanol).-   IR (ν in cm⁻¹): 1600 (C═N), 1632 (C═O), 2926 (CH₂), 2956 (CH₂).-   MS m/e (% B): M⁺218(100), 190(96), 162(25), 132(46), 118(14).-   UV_(ethanol) (in nm) (log ε): 230,4 (4,23), 257,4 (4,37), 305,0    (3,88), 314,6(3,84).-   b) From 1,0 g (4,6 mmol) of the compound prepared in (a),    1-(3-mercaptopropyl)-chinazolin-2,4(1H,3H)-dione is prepared by a    method analogous to that described in Example 1 d.-   Colorless crystals.-   Yield 79%.-   C₁₁H₁₂N₂O₂S (236,3).-   F.: 154°-157° C. (ethanol).-   IR (ν in cm⁻¹): 1610 (NH), 1682 (C═O), 1702 (CH₂), 2544 (SH).-   MS m/e (% B): M⁺236(81), 203(100), 189(10), 176(32), 162(16),    160(26), 146(33), 132(98).-   UV_(ethanol) (in nm) (log ε): 220,4 (4,74), 245,2 (4,13), 313,0    (3,87)

EXAMPLE 5

1-(2-mercaptoethyl)-chinazolin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=R⁴=hydrogen, R³=mercapto, n=1, A=benzene ring withR⁵=hydrogen).

-   a) 1,2-dihydro-5H-thiazolo[3,2-a]chinazolin-5-one (structure V,    R⁷=R⁸=R⁹=R¹⁰=hydrogen, A=benzene ring with R⁵=hydrogen).

The compound, previously described in the literature, is preparedanalogously to Example 4 method (a) with 5,04 g (24 mmol) 2-ammoniumbenzoic acid methyl ester thiocyanate and 50,0 g 1,2-dibromomethane.

-   Colorless crystals.-   Yield 31% based on 2-ammoniumbenzoic add methyl ester thiocyanate.-   C₁₀H₈N₂OS (204,3).-   F.: 238°-239° C. (2-methoxyethanol).-   IR (ν in cm⁻¹): 1608 (C═N), 1644 (C═O), 2948 (CH₂), 2986 (CH₂).-   MS m/e (% B): M⁺204(100), 176(83), 132(38), 104(11).-   UV_(ethanol) (in nm) (log ε): 232,8 (4,13), 254,4 (4,21), 308,8    (3,63).-   b) 1-(2-mercaptoethyl)-chinazolin-2,4(1H,3H)-dione is prepared    analogously to Example 1 (d) from 1,02 g (5,0 mmol) of the compound    prepared in (a).-   Colorless crystals.-   Yield 84%.-   C₁₀H₁₀N₂O₂S (223,3).-   F.: 178°-181° C. (ethanol).-   IR (ν in cm⁻¹): 1606 (NH), 1698 asymm. (C═O), 2552 (SH).-   MS m/e (% B): M⁺222(100).-   UV_(ethanol) (in nm) (log ε): 220,4 (4,43), 245,2 (3,83), 313,0    (3,57).

EXAMPLE 6

1-(2-mercaptoethyl)-3-methylchinazolin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=hydrogen, R³=mercapto, R⁴ methyl, n=1, A=benzenering with R⁵=hydrogen).

1,92 g (10 mmol) of the previously described3-methyl-chinazolin-4(3H)-on-2(1H)-thione (structure VII, R4=methyl,A=benzene ring with R⁵=hydrogen) is reacted in 30 ml absolutedimethylformamide with 1,5 g (11 mmol) dry potassium carbonate. Withconstant stirring, 2,23 g (12 mmol) 1,2-dibromomethane is addeddropwise. After 24 h, 150 ml 10% aqueous hydrochloric acid is added, thereaction mixture refluxed for 8, and finally filtered hot. The filtrateis cooled and held for 12 h at ca. 4° C. The resulting precipitate iswashed with water, dried, and recrystallized. Thin-layerchromatographically pure1-(2-mercaptoethyl)-3-methyl-chinazolin-2,4(1H,3H)-dione (780 mg) isobtained.

-   Colorless crystals.-   Yield 33%.-   C₁₁H₁₂N₂O₂S (236,3).-   F.: 171°-175° C. (ethanol).-   IR (ν in cm⁻¹): 1648 (C═O), 1696 (C═O), 2560 (SH), 2942 (CH₂).-   MS m/e (% B): M⁺236(13), 189(6), 176(100), 146(7), 132(81), 119(34).-   UV_(etanol) (in nm) (log ε): 220,5 (4,60), 248,0 (3,88), 311,0    (3,68).

EXAMPLE 7

1-(3-mercaptoprop-1-yl)-3-methyl-chinazolin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=hydrogen, R³=mercapto, R⁴=methyl, n=2, A=benzenering with R⁵=hydrogen).

Analogous to Example 6, 1,92 g 10 mmol)3-methyl-chinazolin-4(3H)-on-2(1H)-thione is reacted with 2,42 g (12mmol) 1,3-dibromopropane. Thin-layer chromatographically pure1-(2-mercaptoprop-1-yl)₃-methyl-chinazolin-2,4(1H,3H)-dione (525 mg) isobtained.

-   Colorless crystals.-   Yield 21%.-   C₁₂H₁₄N₂O₂S (250,3).-   F.: 105°-107° C. (ethanol).-   IR (ν in cm⁻¹): 1640 (C═O), 1700 (C═O), 2542 (SH), 2932 (CH₂).-   MS m/e (% B): M⁺250(65), 217(83), 203(9), 190(30), 176(13), 160(27),    146(35).

EXAMPLE 8

3-ethyl-1-(2-mercaptoethyl)-chinazolin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=hydrogen, R³=mercapto, R⁴=ethyl, n=1, A=benzenering with R⁵=hydrogen).

Analogous to Example 6, 2,06 g (10 mmol) of the previously described3-ethyl-chinazolin-4(3H)-on-2(1H)-thione is reacted with 2,23 g (12mmol) 1,2-dibromomethane. Thin-layer chromatographically pure3-ethyl-1-(2-mercaptoethyl)-chinazolin-2,4(1H,3H) dione (475 mg) wasobtained.

-   Colorless crystals.-   Yield 19%.-   C₁₂H₁₄N₂O₂S (250,3).-   F.: 114°-115° C. (ethanol).-   IR (ν in cm⁻¹): 1660 (C═O), 1696 (C═O), 2569 (SH).-   MS m/e (% B): M⁺250(11), 203(6), 190(64), 162(41), 146(16),    132(100).

EXAMPLE 9

3-ethyl-1-(3-mercaptoprop-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure a, R¹=R²=hydrogen, R³=mercapto, R⁴=ethyl, n=2, A=benzene ringwith R⁵=hydrogen).

Analogous to Example 6, 2,06 g (10 mmol) of the previously described3-ethyl-chinazolin-4(3H)-on-2(1H)-thione is reacted with 2,42 g (12mmol) 1,2-dibromopropane. Thin-layer chromatographically pure3-ethyl-1-(3-mercaptoprop-1-yl)-chinazolin-2,4(1H,3H)-dione (607 mg) wasobtained.

-   Colorless crystals.-   Yield 23%.-   C₁₃H₁₆N₂O₂SxH₂O (264,3+xH₂O).-   F.: 110°-114° C. (ethanol).-   IR (ν in cm⁻¹): 1652 (C═O), 1702 (C═O), 2562 (SH), 2932 (CH₂).-   MS m/e (% B): M⁺264(55), 231(67), 217(8), 203(16), 176(42), 160(23),    146(45).-   UV^(ethanol) (in nm) (log ε): 222,2 (4,75), 244,0 (4,08), 313,0    (3,87).

EXAMPLE 10

6,7-dimethoxy-1-(2-mercaptoethyl)-3-methyl-chinazolin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=hydrogen, R³=mercapto, R⁴=methyl, n=1, A=benzenering with R⁵ in the 6- and 7-positions each —OCH₃).

Analogous to Example 6, 2,52 g (10 mmol) of the previously described6,7-dimethoxy-3-methyl-chinazolin-4(3H)-on-2(1H)-thione is reacted with2,23 g (12 mmol) 1,2-dibromomethane. Thin-layer chromatographically pure6,7-dimethoxy-1-(2-mercaptoethyl)-3-methyl-chinazolin-2,4(1H,3H)-dione(1,36 g) was obtained.

-   Colorless crystals.-   Yield 46%.-   C₁₃H₁₆N₂O₄S (296,3).-   F.: 121°-122° C. (ethanol).-   IR (ν in cm⁻¹): 1650 (C═O), 1696 (C═O), 2550 (SH), 2936 (CH₂).-   MS m/e (% B): M⁺296(52), 236(100), 221(43), 192(89), 164(18),    149(20), 134(13).-   UV_(ethanol) (in nm) (log ε): 237,0 (4,59), 259,5 (3,95), 322,0    (3,84).

EXAMPLE 11

3-benzyl-1-(2-mercaptoethyl)-chinazolin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=hydrogen, R³=mercapto, R⁴=benzyl, n=1, A=benzenering with R⁵=hydrogen).

Analogous to Example 6, 2,68 g (10 mmol) of the previously described3-benzyl-chinazolin-4(3H)-on-2(1H)-thione is reacted with 2,23 g (12ibmol) 1,2-dibromomethane. Thin-layer chromatographically pure3-benzyl-1-(2-mercaptoethyl)-chinazolin-2,4(1H,3H)-dione (905 mg) wasobtained.

-   Colorless crystals.-   Yield 29%.-   C₁₇H₁₆N₂O₂S (312,4).-   F.: 111°-114° C. (ethanol).-   IR (ν in cm⁻¹): 1656 (C═O), 1698 (C═O), 2573 (SH).-   MS m/e (% B): M⁺312(13), 252(93), 235(7), 146(23), 132(100).-   UV_(ethanol)(in nm) (log ε): 223,0 (4,81), 246.2 (4,15), 311,8    (3,84).

EXAMPLE 12

3-(4-mercaptobut-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=n-butylene [—(CH₂)₄-, X=mercapto, A=benzene ringwith R⁵=hydrogen).

-   a) 3-(4-hydroxybut-1-yl)-chinazolin-4(3H)-on-2(1H)-thione.-   (structure XI, A=benzene ring with R⁵=hydrogen).

9,65 g (0,05 mol) of the previously described 2-isothiocyanate benzoicacid methyl ester (structure X, Alk=methyl, A=benzene ring withR⁵=hydrogen) is reacted with 4,45 g (0,05 mol) 4-aminobutan-1-ol. Thereaction mixture is stirred for 24 h at room temperature, then 40 mlwater is added. The resulting precipitate is collected on a glass frit,washed with water, and after drying, recrystallized to obtain 10,6 g3-(4-hydroxybut-1-yl)-chinazolin-4(3H)-on-2(1H)-thione.

-   Colorless crystals.-   Yield 85%.-   C₁₂H₁₄N₂O₂Sx0,5H₂O (250,3+9,0).-   F.: 168°-172° C. (ethanol).-   IR (ν in cm⁻¹): 1622 (NH), 1652 (C═O), 2950 (CH₂), 3408 (OH).-   MS m/e (% B): M⁺+1 251(100).-   b) 2,3,4,5-tetrahydro-7H-[1,3]thiazepino[2,3-b]chinazolin-7-one    (structure XII, A=benzene ring with R⁵=hydrogen).

5,0 g (0,02 mol) of the thin-layer chromatographically pure compounddescribed in (a) is taken up in 35 ml concentrated hydrochloric acid andrefluxed for 90 min. After cooling, the precipitate is collected byfiltration, and sufficient 5% sodium carbonate is added to obtain pH 8.The precipitate is washed with water, dried, and recrystallized toobtain 3,8 g2,3,4,5-tetrahydro-7H-[1,3]thiazepino[2,3-b]chinazolin-7-one.

-   Colorless crystals.-   Yield 82%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 116°-118° C. (ethanol).-   IR (ν in cm⁻¹): 1604 (C═N), 1668 (C═O), 2960 (CH₂).-   MS m/e (% B): M⁺232(21), 217(100), 199(54), 179(34), 162(69),    146(30), 119(43).-   UV_(ehtanol) (in nm) (log ε): 226,0 (4,3B), 295,5 (4,16).-   c) 3-(4-mercaptobut-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=n-butylene [—(CH₂)₄-], X=mercapto, A=benzene ringwith R⁵=hydrogen).

1,50 g (6,45 mol) of the compound described in (b) is taken up in amixture of 0,6 ml concentrated sulfuric acid, 1,5 ml glacial aceticacid, and 66 ml water and refluxed for 8 h. After cooling, theprecipitate is collected on a glass frit, washed with a small amount ofwater, dried, and recrystallized to obtain 1,43 g3-(4-mercaptobut-1-ylychinazolin-2,4(1H,3H)-dione

-   Colorless crystals.-   Yield 89%.-   C₁₂H₁₄N₂O₂S (250,3).-   F.: 157°-160° C. (ethanol).-   IR (νin cm⁻¹): 1606 (NH), 1664 (C═O), 1712 (C═O), 2573 (SH), 2936    (CH₂).-   MS m/e (% B): M⁺250(11), 217(51), 203(3), 187(4), 163(100), 146(76),    119(35).-   UV_(ethanol) (in nm) (log ε): 219,5 (4,67), 244,0 (3,97), 310,5    (3,65)

EXAMPLE 13

3-(5-mercaptopent-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=n-pentylene [—(CH₂)₅-], X=mercapto, A=benzene ringwith R⁵ hydrogen).

-   a) 3-(5-hydroxypent-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure XV, Alk*=n-pentylene [—(CH₂)₅—], A=benzene ring withR⁵=hydrogen).

6,27 (0,03 mol) of the previously described2-methoxycarbonylaminobenzoic acid methyl ester (structure XIII, Alk=ineach case methyl, A=benzene ring with R⁵=hydrogen) is refluxed with 5,15g (0,05 mol) 5-aminopentan-1-ol (structure XIV, Alk*=n-pentylene[—(CH₂)₅-]) for 10 min with moderate heating. After cooling, 100 mlwater is added with stirring, and finally sufficient dilute hydrochloricacid is added to ca. pH 4. The crude product is crystallized by theaddition of a small amount of ethanol to yield, after filtration anddrying, 6,18 g 3-(5-hydroxypent-1-yl)-chinazolin-2,4(1H,3H)-dione.

-   Colorless crystals.-   Yield 83%.-   C₁₃H₁₆N₂O₃x1H₂O (248,3+18,0).-   F.: 125°-129° C. (ethanol/water).-   IR (νin cm⁻¹): 1632 asymm. (NH, C═O), 1720 (C═O), 2936 (CH₂).-   MS m/e (% B): M⁺248(10), 231(8), 218(20, 176(35, 163(100), 146(77),    119(56).-   b) 3-(5-bromopent-1-yl)-chinazolin-2,4(1H,3)-dione

(structure XVI, Alk*=n-pentylene [—(CH₂)₅-], Hal=bromine, A=benzene ringwith R⁵=hydrogen).

2,48 g (0,01 mol) of the thin-layer chromatographically pure compoundprepared in (a) is taken up in 25 ml concentrated hydrobromic acid andheated to reflux for 30 min. After cooling, the precipitate is collectedby filtration and dried to yield 2,89 g thin-layer chromatographicallypure 3-(5-bromopent-1-yl)-chinazolin-2,4(1H,3H)-dione.

-   Colorless needles.-   Yield 93%.-   C₁₃H₁₅N₂O₂Br (311,2).-   F.: 169°-172° C. (ethanol).-   IR (ν in cm⁻¹): 1622 (NH), 1666 (C═O), 1712 (C═O), 2940 (CH₂).-   MS m/e (% B): M⁺+1 313(96,311(100).-   c) 3-(5-mercaptopent-1-yl)-chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=n-pentylene [—(CH₂)₅-], X=mercapto, A=benzene ringwith R⁵=hydrogen).

1,55 g (5,0 mmol) of the compound prepared in (b) is taken up in 5 ml2-methoxyethanol, to which is added 0,46 g (6 mmol) thiourea. Thereaction mixture is refluxed for 30 min. After cooling, 50 ml water isadded and sufficient 4% aqueous sodium hydroxide to achieve alkalinepH., After the solution clears, it is filtered and then acidified with10% aqueous hydrochloric acid. The precipitate is collected, washed, anddried to yield 780 mg3-(5-mercaptopent-1-yl)-chinazolin-2,4(1H,3H)-dione.

-   Colorless crystals.-   Yield 59%.-   C₁₃H₁₆N₂O₂S (264,3).-   F.: 137°-138° C. (ethanol).-   IR (ν in cm⁻¹): 1636 asymm. (NH, C═O), 1726 (C═O), 2585 (SH), 2936    (CH₂).-   MS m/e (% B): M+264(9), 231(46), 176(10), 163(100), 146(48),    119(26).-   UV_(ethanol) (in nm) (log ε): 220,0 (4,91), 243,5 (4,23), 310,8    (3,93).

EXAMPLE 14

1-(3-mercaptoprop-1-yl)-3-methyl-thieno(3,2-d)-pyrimidin-2,4(1H,3H)-dione

(structure Ia, R¹=R²=hydrogen, R³ mercapto, R⁴=methyl, n=2,A=2,3-annealed thiophene ring).

Analogous to example 6, 0,99 g (5 mmol) of the previously described3-methyl-thieno[3,2d]-pyrimidin-4(3H)-on-2(1H)-thione is reacted with1,11 g (5,5 mmol) 1,3-dibromopropane and 0,75 g (5,4 mmol) potassiumcarbonate in 15 ml absolute dimethylformamide. After 2 h, 60 ml 10%hydrochloric acid is added and the mixture refluxed for an additional 3h to yield 1,14 g thin-layer chromatographically pure1-(3-mercaptoprop-1-yl)-3-methyl-thieno(3,2-d)-pyrimidin-2,4(1H,3H)-dione.

-   Ochre crystals.-   Yield 89%.-   C₁₀H₁₂N₂O₂S₂ (264,3).-   F.: 174°-176° C. (ethanol).-   IR (ν in cm⁻¹): 1660 (C═O), 1691 (C═O), 2560 (SH).-   MS m/e (% B): M⁺256(100), 223(50), 196(39), 152(40), 138(93),    125(23), 110(24).-   UV_(ethanol) (in nm) (log ε): 219,6 (0,42), 273,7 (3,23), 306,2    (3,42).

EXAMPLE 15

Tablets with 50,0 mg1-(3-mercaptoprop-1-yl)-3-methyl-chinazolin-2,4(1H,3H)-dione

-   Components:

1 tablet contains 50,0 active substance, 32,0 mg microcrystallinecellulose, 20,0 mg lactose, 15, 0 mg potato starch, 8,0 mg talc, 3,2 mgpolyvinylpyrrolidone, and 1,5 mg magnesium stearate.

Preparation:

The powdered active substance is mixed to homogeneity withmicrocrystalline cellulose, lactose, and potato starch in a 20% w/vsolution of polyvinylpyrrolidone in ethanol. The moist mass is pressedthrough a 1,5-mm mesh seive, dried at 40° C., and again pressed througha 1,0-mm mesh seive. The resulting granulate is mixed with talc andmagnesium stearate and pressed into tablets.

-   Tablet weight: 130 mg.

EXAMPLE 16

Coated Tablets with 35 mg1-(3-mercaptoprop-1-yl)-chinazolin-2,4(1H,3H)-dione

-   Components:

One coated tablet contains 35 mg active substance, 20,0 mgmicrocrystalline cellulose, 10,0 mg lactose, 16,5 mg cornstarch, 5,0 mgtalc, 2,8 mg polyvinylpyrrolidone, and 0,7 mg magnesium stearate.

Preparation:

The powdered active substance is mixed to homogeneity withmicrocrystalline cellulose, lactose, and cornstarch in a 20% w/vsolution of polyvinylpyrrolidone in ethanol. The moist mass is pressedthrough a 1,5-mm mesh seive, dried at 40° C., and again pressed througha 1,0-mm mesh seive. The resulting granulate is mixed with talc andmagnesium stearate and pressed into tablet cores.

-   Core weight: 90 mg.

The tablet or suppository cores as described are coated and polished bystandard methods.

EXAMPLE 17

1-(2-mercaptoethyl)-3-methyl-thieno[3,2-d]-pyrimidine-2,4(1H,3H)-dione

(structure Ia, R¹, R²=hydrogen, R³=mercapto, R⁴=methyl, n=1,A=2,3-annealed thiophene ring).

Analogous to example 14, 0,99 g (5 mmol) of the previously described3-methyl-thieno[3,2-d]-pyrimidin-4(3H)-on-2(1H)-thione is reacted with1,03 g (5,5 mmol) 1,2-dibromomethane to produce 420 mg thin-layerchromatographically pure1-(2-mercaptoethyl)-3-methyl-thieno[3,2-d]-pyrimidine-2,4(1H,3H)-dione.

-   Ochre crystals.-   Yield 41%.-   C₉H₁₀N₂O₂S₂ (242,3).-   F.: 158°-160° C. (ethanol).-   IR (ν in cm⁻¹): 1648 (C═O), 1692 (C═O), 2590 (SH).-   MS m/e (% B): M⁺242(27), 183(83), 138(100), 125(32), 110(18).-   UV_(ethanol) (in nm) (log ε): 222,5 (4,20), 270,8 (2,78), 316,7    (3,05).

EXAMPLE 18

(R,S)-3-[3-(1-methyl)-mercaptopropyl]-chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=1-methyl-propyl, X=3-mercapto, A=annealed benzenering with R⁵=hydrogen).

-   a) 2-crotylthiochinazolin-4(3H)-one

The previously described compound is prepared in thin-layerchromatographically pure form from 3,56 g (20 mmol)chinazolin-4(3H)-on-2(1H)-thione and 1,81 g (20 mmol) crotyl chloride.

-   Colorless crystals.-   Yield 71%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 193°-194° C. (ethanol).-   IR (ν in cm⁻¹): 1613 (C═N), 1673 (C═O), 2953 (CH₂).-   MS m/e (% B): M⁺+1 233(100).-   UV_(ethanol) (in nm) (log ε): 230,5 (4,47), 274,5 (4,24), 314,5    (3,84).-   b)    (R,S)-4-methyl-3,4-dihydro-2H,6H-[1,3]thiazino[2,3-b]chinazolin-6-one    1,15 g (5 mmol) of the compound obtained in (a) is taken up in 5 ml    concentrated sulfuric acid and stirred for 2 h at 40° C. After this,    100 ml ice-cold water is added and saturated sodium carbonate, and    if necessary 1N aqueous sodium hydroxide, is added to neutrality.    The resulting precipitate is collected, dried, and recrystallized.-   Colorless crystals.-   Yield 51%.-   C₁₂H₁₂N₂OS (232,3).-   F.: 111°-112° C. (ethanol).-   IR (ν in cm⁻¹): 1608 (C═N), 1678 (C═O), 2938 (CH₂).-   MS mile (% B): M⁺²³²(100).-   UV_(ethanol)(in nm) (log ε): 235,0 (4,32), 283,8 (4,15).-   c) (R,S)-3-[3-(1-methyl)-mercaptopropyl]-chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=1-methyl-propyl, X=3mercapto, A=annealed benzenering with R⁵=hydrogen).

Analogous to example 1, step 1d, 0,5 g (2 mmol) thiazo-compound (b) istaken up in the acid mixture, heated to reflux, and finallyrecrystallized.

-   Colorless crystals.-   Yield 71%.-   C₁₂H₁₂N₂O₂S (232,3).-   F.: 114°-118° C. (ethanol).-   IR (ν in cm⁻¹), 1620 (NH), 1644 (C═O), 1720 (C═O), 2562 (SH).-   MS m/e (% B): M⁺250(11), 232(13), 217(96), 201(14), 163(100),    146(79), 119(26), 90(29).-   UV_(ethanol) (in nm) (log ε): 220,0 (4,67), 242,0 (4,10), 313,0    (3,87).

EXAMPLE 19

(R,S)-3-[3-(2-methyl)-mercaptopropyl]chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=2-methyl-propyl, X=3-mercapto, A=annealed benzenering with R⁵=hydrogen).

-   a)    (R,S)-3-methyl-3,4-dihydro-2H,6H-[1,3]thiazino[2,3-b]-chinazolin-6-one    3,65 g (20 mmol) of the previously described compound    chinazolin-4(3H)-on-2(1 H)-thione and 2,6 g (20 mmol) potassium    carbonate are mixed in 30 ml dimethylformamide. After dissolution of    the chinazoline, 2,19 g (22 mmol) 1-chloro-3-bromo-2-methylpropane    is added and the mixture shaken for 2 days. The precipitate is    recovered and recrystallized.-   Colorless crystals.-   Yield 79%.-   C₁₂H₁₂N₂OSxH₂O (232,3+xH₂O).-   F.: 141°-146° C. (ethanol).-   IR (ν in cm⁻¹): 1606 (C═N), 1682 (C═O), 2932 (CH₂).-   MS m/e (% B): M⁺232(100), 217(38), 199(26), 162(46).-   UV_(ethanol)(in nm) (log ε): 237,0 (4,28), 290,5 (4,25).-   b) (R,S,)-3-(3-(2-methyl(-mercaptopropyl]chinazolin-2,4(1H,3H)-dione

(structure Ib, Alk*=2-methyl-propyl, X=3-mercapto, A=annealed benzenering with R⁵=hydrogen).

Analogous t6 example 1, step 1d, 0,5 g of the compound described in (a)was used.

-   Colorless crystals.-   Yield 79%.-   C₁₂H₁₂N₂OSxH₂O (232,3+xH₂O).-   F.: 194°-197° C. (ethanol).-   IR (ν in cm⁻¹): 1640 asymm. (NH, C═O), 1722 (C═O), 2572 (SH).-   MS m/e (% B): M⁺250(24), 217(100), 201(17), 176(43), 163(57),    146(84), 119(47), 90(25).-   UV_(ethanol) (in nm) (log ε): 219,4 (4,66), 240,0 (4,12), 293,0    (3,99).

EXAMPLE 20

N-hydroxy-2-[3-(methyl-2,4-dioxo-3,4-dihydro-2H-chinazolin-1-yl)-propylsulfanyl]acetamide

(structure Ia, R¹=R²=hydrogen, R³=hydroxycarbamoylmethylenethio,R⁴=methyl, n=2, A=benzene ring with R⁵=hydrogen).

250 mg (1 mmol) 1-(3-mercaptoprop-1-yl)-3-methyl-chinazolin-2,4(1H,3Hdione (as in example 7) is dissolved in 4 ml absolute dimethylformamideunder nitrogen. Then, 330 mg (3 mmol) 2-chloro-N-hydroxyacetamide and0,83 ml (6 mmol) triethylamine are added and the mixture stirred undernitrogen for 3 h. After the addition of 1 ml water, on ice, the mixtureis acidified with glacial acetic acid and stirred at 0° C. for anadditional 2 h. The precipitated solid is collected, washed with water,dried, and recrystallized.

-   Colorless crystals.-   Yield 66%.-   Cl₄H₁₇N₃O₄S (323,4).-   F.: 146°-148° C. (ethyl acetate/petroleum ether).-   IR (ν in cm⁻¹): 1488 (C═O), 1646 (C═O), 1697 (C═O), 3200-3500 (OH).-   MS m/e (% B): M⁺323(5), 264(5), 250(50), 217(35).-   UV_(ethanol) (in nm) (log ε): 215,4 (4,26), 302,1 (3,04), 332,9    (2,71).

1. A method of inhibiting collagenase/MMP activities in a mammalcomprising administering to the mammal in need thereof an effectiveamount of one or more polycyclic (pyrimidine-2,4(1H, 3H)-diones) withfunctionalized alkyl groups in the 1-, 3-, or both positions with thegeneral structures Ia and Ib,

where: R¹ is hydrogen, methyl, or ethyl; R² is hydrogen or methyl; R³ ismercapto or hydroxyaminoacylalkylthio (—SAlkCONHOH); Alk is C₁-C₅branched or unbranched alkyl; R⁴ is hydrogen, benzyl, or phenyl; n is 0,1 or 2; Alk* is C₂-C₁₂ branched or unbranched alkylene, with theexception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)-]; X is mercapto orhydroxyaminoacylalkylthio (—SAlkCONHOH); A is an annealed benzene ringor a 2,3-annealed thiophene ring, wherein the 4,5-positions areoptionally substituted with methyl groups or are optionally annealedwith a cyclopentene, cyclohexene, or cycloheptene ring, R⁵ is hydrogen,6-methyl, 8-methyl, 6-fluoro, 6-choloro, 6-bromo, 6-methylthio, or6,7-dimethoxy, as well as the tautomers and pharmacologically relevantsalts of these compounds.
 2. A method of inhibiting tumor metastasis andinvasion in a mammal comprising administering to the mammal in needthereof an effective amount of one or more polycyclic(pyrimidine-2,4(1H, 3H)-diones) with functionalized alkyl groups in the1-, 3-, or both positions with the general structures Ia and Ib,

where: R¹ is hydrogen, methyl, or ethyl; R² is hydrogen or methyl; R³ ismercapto or hydroxyaminoacylalkylthio (—SAlkCONHOH); Alk is C₁-C₅branched or unbranched alkyl; R⁴ is hydrogen, benzyl, or phenyl; n is 0,1 or 2; Alk* is C₂-C₁₂ branched or unbranched alkylene, with theexception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—], X is mercapto orhydroxyaminoacylalkylthio (—SAlkCONHOH); A is an annealed benzene ringor a 2,3-annealed thiophene ring, wherein the 4,5-positions areoptionally substituted with methyl groups or are optionally annealedwith a cyclopentene, cyclohexene, or cycloheptene ring, R⁵ is hydrogen,6-methyl, 8-methyl, 6-fluoro, 6-choloro, 6-bromo, 6-methylthio, or6,7-dimethoxy, as well as the tautomers and pharmacologically relevantsalts of these compounds.
 3. A method of treating UV-induced erythema ina mammal comprising administering to the mammal in need thereof aneffective amount of one or more polycyclic (pyrimidine-2,4(1H,3H)-diones) with functionalized alkyl groups in the 1-, 3-, or bothpositions with the general structures Ia and Ib,

where: R¹ is hydrogen, methyl, or ethyl; R² is hydrogen or methyl; R³ ismercapto or hydroxyaminoacylalkylthio (—SAlkCONHOH); Alk is C₁-C₅branched or unbranched alkyl; R⁴ is hydrogen, benzyl, or phenyl; n is 0,1 or 2; Alk* is C₂-C₁₂ branched or unbranched alkylene, with theexception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]; X is mercapto orhydroxyaminoacylalkylthio (—SAlkCONHOH); A is an annealed benzene ringor a 2,3-annealed thiophene ring, wherein the 4,5-positions areoptionally substituted with methyl groups or are optionally annealedwith a cyclopentene, cyclohexene, or cycloheptene ring, R⁵ is hydrogen,6-methyl, 8-methyl, 6-fluoro, 6-choloro, 6-bromo, 6-methylthio, or6,7-dimethoxy, as well as the tautomers and pharmacologically relevantsalts of these compounds.
 4. A method of treating rheumatic diseases ina mammal comprising administration to the mammal in need thereof aneffective amount of one or more polycyclic (pyrimidine-2,4(1H,3H)-diones) with functionalized alkyl groups in the 1-, 3-, or bothpositions of the general structures Ia and Ib,

where: R¹ is hydrogen, methyl, or ethyl; R² is hydrogen or methyl; R³ ismercapto or hydroxyaminoacylalkylthio (—SAlkCONHOH); Alk is C₁-C₅branched or unbranched alkyl; R⁴ is hydrogen, benzyl, or phenyl; n is 0,1 or 2; Alk* is C₂-C₁₂ branched or unbranched alkylene, with theexception of 3-methylpropylene [—CH₂—CH₂—CH(CH₃)—]; X is mercapto orhydroxyaminoacylalkylthio (—SAlkCONHOH); A is an annealed benzene ringor a 2,3-annealed thiophene ring, wherein the 4,5-positions areoptionally substituted with methyl groups or are optionally annealedwith a cyclopentene, cyclohexene, or cycloheptene rine, R⁵ is hydrogen,6-methyl, 8-methyl, 6-fluoro, 6-choloro, 6-bromo, 6-methylthio, or6,7-dimethoxy, as well as the tautomers and pharmacologically relevantsalts of these compounds.
 5. The method of claim 1, wherein the one ormore polycyclic (pyrimidine-2,4(1H, 3H)-diones) are compounds of thegeneral structures IIa and IIb

where R¹, R², R³, R⁴, R⁵, Alk, Alk*, n, and X are defined as in claim 1,including their tautomers and pharmacologically relevant salts.
 6. Themethod of claim 2, wherein the one or more polycyclic(pyrimidine-2,4(1H, 3H)-diones) are compounds of the general structuresIIa and IIb

where R¹, R², R³, R⁴, R⁵, Alk, Alk*, n, and X are defined as in claim 2,including their tautomers and pharmacologically relevant salts.
 7. Themethod of claim 3, wherein the one or more polycyclic(pyrimidine-2,4(1H, 3H)-diones) are compounds of the general structuresIIa and IIb

where R¹, R², R³, R⁴, R⁵, Alk, Alk*, n, and X are defined as in claim 3,including their tautomers and pharmacologically relevant salts.
 8. Themethod of claim 4, wherein the one or more polycyclic(pyrimidine-2,4(1H, 3H)-diones) are compounds of the general structuresIIa and IIb

where R¹, R², R³, R⁴, R⁵, Alk, Alk*, n, and X are defined as in claim 4,including their tautomers and pharmacologically relevant salts.